Polyacrylamide Gel Electrophoresis (PAGE) is a protein analysis tool, extremely useful for separation, identification, purity verification, and size determination of proteins. Particularly, SDS-PAGE (Sodium Dodecyl Sulfate-PAGE) is a method wherein a protein is strongly bound to an anionic surfactant, i.e. SDS, at the ratio of 1.4:1 to have (−) charge on the surface, and so only its size acts as a separation factor. This method is widely used for protein analysis because it can be easily handled and has a good resolution rate (A. T. Andrews, Electrophoresis, 1-58).
Since most proteins that are subjects of analysis are colorless, and so an appropriate means is required for their detection. Therefore, various detection methods, e.g. organic dye staining method, silver staining method, fluorescence staining method, background staining method, etc., have been reported hitherto.
Organic dye staining method detects proteins by staining protein bands with an organic dye, e.g. Amido Black 10B, Ponceaus S, Fast Green FCF, Coomassie Brilliant Blue R (CBBR), etc. [J. of Chromatography A, 698, 123-143 (1995)]. Particularly, since the CBBR staining method is relatively simple and cost-effective, it has been generally used. However, the CBBR method has the problems that it takes a long time (4 to 6 hours) for staining and destaining, and does not have such good sensitivity [50 ng for Bovine Serum Albumin (BSA)].
Silver staining method, which has the highest sensitivity among non-radiological detection methods, is a method to impregnate and reduce silver ions [J. of Chromatography A, 698, 123-143 (1995)]. This method has very high sensitivity, capable of detecting proteins of 0.1 ng, but has such problems as the handling is complicated and the process is multi-step. It can be classified into silver nitrate method and diamine silver method depending on how to impregnate and reduce silver ions. Generally, the diamine silver method has good sensitivity, but requires complicated steps, and so the silver nitrate method is more widely used. However, in the silver nitrate method, an oxidizing agent like dichromate, or a reducing agent like glutaraldehyde or DTT must be used to overcome its low sensitivity and high background. Employment of such substances improves detection sensitivity, but may cause protein modifications or be harmful to a handler [Electrophoresis 13, 429-439 (1992)].
On the other hand, fluorescence staining method detects proteins by labeling the proteins with a fluorescent dye. This method has high detection sensitivity, but has the drawbacks to require UV irradiation and accurate quantitative instrumentation, and thereby to increase costs.
Background staining method, applicable only to an SDS-containing gel, detects proteins by forming precipitates with a metal salt on the gel surface excluding protein bands. This method can detect proteins within a short period of time, and so its utility is great. However, it also has a problem that storing the stained gel is difficult because the resulting band is not persistent [Anal. Biochem. 174, 157-167 (1988)].
Therefore, there has been a need to develop a new method for detecting proteins on polyacrylamide gels, which can rapidly and easily detect the proteins with high sensitivity as compared with the known staining methods.